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Induction of a 26-kDa membrane-form tumor necrosis factor (TNF)-alpha in human alveolar macrophages
1Third Department of Internal Medicine, University of Tokushima, School of Medicine, Japan.
Abstract:
The expressions of a membrane form of TNF (m-TNF) by human alveolar macrophages (AM) and autologous blood monocytes from healthy donors were examined. Upon lipopolysaccharide (LPS) stimulation, AM produced 26-kDa TNF-alpha on their cell surface. We designed a bioassay for measuring m-TNF in which macrophages were fixed with paraformaldehyde after stimulation for 18 h, then m-TNF activity was assessed as cytotoxicity of fixed macrophages on L929 cells. This assay was specific to m-TNF because: 1) no soluble factors were contributed to the cytotoxicity of fixed AM, 2) anti-TNF-alpha monoclonal antibody completely neutralized m-TNF activity, and 3) m-TNF activity was not altered after low-pH or high-salt treatment. On LPS stimulation, AM produced significant amounts of m-TNF earlier than TNF-alpha secretion. AM also expressed significant amounts of m-TNF when stimulated with other bacterial components and their derivatives. Interleukin (IL)-4 suppressed both m-TNF production and TNF-alpha secretion. p-Toluene-sulfonyl-L-arginine methyl ester (TAME) inhibited specifically TNF-alpha secretion and not m-TNF expression. Although blood monocytes produced small amounts of m-TNF, monocyte-derived macrophages showed enhanced m-TNF after cultivation with GM-CSF for 10 days. These findings indicate that m-TNF is expressed as a step in the TNF-alpha producing system, and suggest that m-TNF may play important roles in exhibition of macrophage function in situ.
Insights
Human alveolar macrophages express membrane TNF-alpha (m-TNF) early upon stimulation. This study developed a bioassay to measure m-TNF, revealing its role in the TNF-alpha production pathway and potential in situ macrophage function.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Tumor Necrosis Factor-alpha (TNF-alpha) is a key inflammatory cytokine.
- Membrane-bound TNF-alpha (m-TNF) is the precursor to soluble TNF-alpha.
- Understanding m-TNF expression is crucial for inflammatory response research.
Purpose of the Study:
- To investigate the expression of m-TNF by human alveolar macrophages (AM) and monocytes.
- To develop and validate a bioassay for measuring m-TNF activity.
- To elucidate the role of m-TNF in the TNF-alpha production pathway.
Main Methods:
- Human AM and monocytes were stimulated with lipopolysaccharide (LPS) and other bacterial components.
- A novel bioassay was developed using paraformaldehyde-fixed macrophages to measure m-TNF cytotoxicity.
- Specificity of the m-TNF assay was confirmed using monoclonal antibodies and varying pH/salt conditions.
Main Results:
- AM expressed significant m-TNF on their surface earlier than secreting soluble TNF-alpha upon LPS stimulation.
- The developed bioassay specifically measured m-TNF activity, distinct from soluble factors.
- Interleukin-4 (IL-4) suppressed both m-TNF and TNF-alpha, while TAME selectively inhibited TNF-alpha secretion.
Conclusions:
- m-TNF is expressed as an early step in the TNF-alpha production system by macrophages.
- m-TNF may play a significant role in macrophage function in vivo.
- Monocyte-derived macrophages show enhanced m-TNF expression after GM-CSF cultivation.